Methods and Apparatus for Fabricating Porous Three-Dimensional Tubular Scaffolds

ABSTRACT

Disclosed herein are three-dimensional porous tubular scaffolds for cardiovascular, periphery vascular, nerve conduit, intestines, bile conduct, urinary tract, and bone repair/reconstruction applications, and methods and apparatus for making the same.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/220,424, filed Jun. 25, 2009, the content of which is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Regeneration of cellular tissue and organs is a rapidly growing field.Obstacles commonly found with conventional transplantation, such asdonor tissue/organ shortage and host rejection, may be overcome byenabling the host to utilize his or her own cells to regenerate cellulartissue, repair a damaged nerve or blood vessel, etc.

One important approach towards creating engineered tissue is TissueEngineering, which uses a combination of a three-dimensional (3D)scaffold, progenitor cells and biological factors to “grow” a livingtissue or organ. A 3D scaffold provides a structure for the cells toadhere to during their regeneration process, thereby guiding the cellsinto the size and shape of the desired structure. In this tissueregeneration process, the 3D scaffolds must allow cells to readily toattach, rapidly multiply and form new tissue. In the orchestratedprocess of tissue morphogenesis, cells are significantly influenced byboth the micro-architecture of 3D scaffolds and surface biochemicalproperties for their adhesion, proliferation, migration anddifferentiation. Therefore, 3D scaffolds should be designed with apreferred biological property and internal architecture in mind, whereinporosity and material connectivity provide the required structuralintegrity, mass transport and comprehensive micro-environment for celland tissue growth. In addition, cell survival and proliferation withinthe 3D scaffold require access to oxygen and vital molecules. Thedelivery of vital molecules into the 3D scaffold is governed byscaffold-designed structural and topological configuration defined by afew key design parameters, such as, porosity, pore interconnectivity,tortuosity, scaffold material permeability and diffusivity.

The mechanical property of the 3D scaffolds also is an very importantfeature. The mechanical properties of the 3D scaffolds can be designedto meet the application requirement by designing the porous structure orarchitecture of the scaffolds so that the 3D scaffolds can beexpandable, stretchable, or bendable. Some of the applications will needthe cell seeded 3D scaffold to be cultured under cyclic loading to mimicthe in vivo dynamic tissue growth environment. Some 3D scaffolds willalso need to be foldable or compressed to make them easier for delivery.Once these folded scaffolds are delivered to the sites, they can beunfolded or expanded to perform their desired function.

For example a vascular stent must be expandable and stretchable andstrong enough to keep the blood vessel open to allow blood to flowthrough, and thus requires a scaffold to have a design structure toallow for compressing, expanding and bending. The mechanical propertiesof the stent can further be adjusted by using materials with differentmechanical properties.

Human tissue and organ have various sizes and shapes. Many tissue ororgans have distinct tubular structures, such as blood vessels, theesophagus, intestines, the bile conduct, urinary tract, etc. When bloodvessels are damaged and need to be regenerated, a three-dimensionaltubular scaffold is needed for blood vessel regeneration. The scaffoldsprovide the cells with an exogenous skeleton for the cells to adhere andgrow. Therefore, it is important to have a 3D scaffold manufacturingmethod to produce scaffolds which match the size and shape of thetargeted tubular tissue and organ.

A tubular structure implant is also needed when treating some vasculardiseases. For example, a 3D scaffold in the form of a vascular stent canbe used to keep the clogged blood vessel open. Although metallic stentsare currently widely used clinically, biodegradable polymer stents areunder extensive research and development. The greatest advantage ofbiodegradable stent is that the stent will disappear after a period oftime when there is no longer a need to have it in place.

Scaffolds may be manufactured by Rapid Prototyping (RP), a technologythat produces models and prototype parts from 3D computer-aided design(CAD) model data and model data created from 3D object digitizingsystems. Rapid prototyping is a material addition process in whichmaterial is added or deposited at desired location to form the object.Rapid prototyping technologies has also been explored for thedevelopment of manufacturing approaches to create surgical implantmodels for orthopedic and craniofacial surgical procedures. RP systemsprovide possibilities in fabricating porous 3D object with wellcontrolled channels or pores. However, the current RP systems have onlythree axis (XYZ) and are not efficient systems for fabricating tubularporous structures. When fabricating tubular porous structures, thecurrent RP systems often need to use a supporting material which willhave to be removed after fabrication.

In contrast to the RP of material addition process, there is a way offabricating porous polymer tubing or a stent using a material reductionprocess, that is to use a laser tubular micromachining or a lasercutting/removal system that is similar to the fabrication method of ametal vascular stent. In this method, a polymer tube is produced first,and followed by selected removal of polymer in certain areas, accordingto the porous pattern design, using a laser beam. However, in thisfabrication method, a polymer tube has to be made first, followed bylaser cutting. Overall, this is a time consuming two-step process. Also,most of the polymer material is removed or wasted, particularly in thecase of fabrication of a highly porous polymer stent. This fabricationprocess can be expensive as medical grade polymer materials aretypically expensive. Further, the removal or the evaporation of thepolymer by laser beam heating causes toxic gas emission, resulting inair pollution.

Therefore, there is a need to have a one step process or a system whichcan directly produce porous tubular scaffolds from raw polymer materialsbased on a CAD design, which will overcome the drawbacks of a lasercutting system, such as wasting of materials, time consuming and airpolluting, as well as being able to overcome the drawbacks of a RPsystem, in which a supporting material has to be used and removed afterfinishing the fabrication process.

The inventors of the present application have achieved this goal with a4 axis RP system.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide methods tofabricate porous polymeric three-dimensional (3D) tubular scaffoldswhereby the porosity, pore size, structure, and mechanical propertiescan be easily controlled or manipulated.

It is a further object to the present invention to provide a 4 axis RPsystem in which the 4th axis is a computer controlled rotation shaftadded to an xyz position system. In preferred embodiments, the 4 axis RPsystem also has a material delivery system that directly depositspolymer material in a hot melt filament form or a viscous solutionfilament form. The deposited filament adheres to the surface of therotation shaft or bonds to previously extruded filaments that arealready attached to the rotation shaft. In addition, preferredembodiments allow for the fiber diameter to be changed by either varyingthe rotation speed of the 4th axis where the polymer filament isattached, or by varying the XY axis traveling speed, similar to a hotmelt drawing process. This system is particularly suitable for making 3Dtubular scaffolds with complicated micro-porous structures.

Thus, in certain embodiments, the present invention is directed to anapparatus for manufacturing a 3D tubular scaffold comprising: (i) athree-axis XYZ system connected to a base; (ii) a dispensing systemconnected to the XYZ system; (iii) a nozzle connected to the dispensingsystem; and (iv) a fourth axis system comprising a rotary rod or shaftconnected to the base under the nozzle, wherein either the rotary rod,the nozzle or both are capable of moving along a longitudinal axis.

In other embodiment, the present invention is directed to a method ofmaking a 3D tubular scaffold comprising: (i) adding a polymer into theapparatus described herein; and (ii) dispensing the polymer onto arotary rod.

In further embodiment, the present invention is directed to a 3D tubularscaffold comprising struts and/or fibers joined in a porousthree-dimensional pattern, the scaffold having an average pore size fromabout 1 to about 10000 microns.

In further embodiment, the present invention is directed to a specialtubular scaffold called stents which are comprising struts and/or fibersjoined in a pre-designed three-dimensional pattern.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a depicts a micro single screw extruder in certain embodiments ofthe apparatus of the present invention.

FIG. 1 b depicts a hot melt chamber which uses compressed air forextrusion of polymer in certain embodiments of the apparatus of thepresent invention.

FIG. 2 depicts an embodiment of the apparatus used to manufacture 3Dtubular scaffolds of the present invention.

FIGS. 3 (a) and (b) depict an embodiment of the apparatus of the presentinvention, wherein the nozzle moves in a longitudinal direction alongthe rotating shaft.

FIG. 4 depicts polymers of an embodiment of the 3D tubular scaffold ofthe present invention after expansion.

FIG. 5 depicts polymers of an embodiment of the 3D tubular scaffold ofthe present invention after stretching.

FIG. 6 depicts polymers of an embodiment of the 3D tubular scaffold ofthe present invention after a combination of expansion and stretching.

FIG. 7 depicts an embodiment of the 3D tubular scaffold of the presentinvention illustrating an expandable polymer stent.

FIG. 8 depicts another example of a polymer stent.

DETAILED DESCRIPTION

In the 4 axis RP system of the present invention, the 4^(th) axis is acomputer controlled rotation shaft added to an xyz position system. The4 axis RP system also has a material delivery system that directlydeposits polymer material in a hot melt filament form or a viscoussolution filament form. The deposited filament adheres to the surface ofthe rotation shaft or bonds to previously extruded filaments thatalready attached to the rotation shaft, therefore eliminating the needto use glue or controlled heating, as previously described in, e.g. USpatent application (US 20030211130A1 by Sanders et al). In addition, thefiber diameter can be changed by either varying the rotation speed ofthe 4th axis where the polymer filament is attached, or by varying theXY axis traveling speed, similar to a hot melt drawing process. Thissystem is particularly suitable for making 3D tubular scaffolds withcomplicated micro-porous structures.

3D tubular scaffolds made by the 4 axis RP system provides an internaland external space for cellular interactions. The 3D tubular scaffold iscomposed of polymer fibers which are joined together in a pre-designfashion or pattern. Such a configuration allows the tubular scaffold tohave 100% pore interconnection. In addition to the 3D tubular scaffoldand 4 axis RP system, the present invention is also directed to methodsof manufacturing the 3D tubular scaffold and applications for the 3Dtubular scaffold.

3D Tubular Scaffolds

The 3D tubular scaffold of the present invention may be configured inany size to accomplish the particular purpose at hand, e.g., sizesuitable for use in vasculogenesis, osteogenesis, vascular stents, etc.

The 3D tubular scaffolds can be single layered or multi-layered.

The fibers or the struts of the 3D tubular scaffolds may be joinedtogether in a pre-designed fashion or pattern. For example, the fibersmay be joined together in a perpendicular angle)(90°), an acute angle(less than 90°) or at an obtuse angle (more than 90°), or a combinationthereof. Additionally, the fibers of the 3D tubular scaffolds may haveconstant diameters or different diameters. In preferred embodiments, thediameter of the polymer fibers of the 3D tubular scaffolds are fromabout 50 nm to 2 mm, more preferably from 100 μm to 1000 μm.

The cross sections of the struts and/or fibers may be a circle,triangle, square, rectangle, star, or irregular shape.

The pores of the 3D tubular scaffolds may be a constant size and/ordimension, a variable size and/or dimension or a combination thereof.For example, pores may be a constant size and/or dimension within eachlayer, but differ from the pore size and/or dimension on differentlayers. In preferred embodiments, the mean pore size of the 3D tubularscaffolds are from having an average pore size of from about 15 micronsto about 1000 microns, from about 25 microns to about 500 microns, orfrom about 50 microns to about 100 microns. In yet other embodiments, adense layer may be developed without pores. The 3D tubular scaffolds mayhave a pore distribution of 0%, greater than about 50%, greater thanabout 80% or greater than about 95%.

The surface area, porosity and pore size of the 3D tubular scaffolds aredetermined by the design of the constructs, including the size andgeometry of the fibers, number of the fibers in each unit volume and theconstruction pattern of the fibers in the 3D tubular scaffold. Inpreferred embodiments, these factors are further controlled by themobility of certain aspects of the manufacturing apparatus describedherein, namely the rotary rod and/or the nozzle.

The 3D tubular scaffolds of the present invention may be made ofnon-biodegradable polymer, biodegradable polymer, or a combinationthereof.

Non-biodegradable polymers for use in the present invention include, forexample, non-degradable synthetic polymers, e.g., polystyrene,polyethylene, polypropylene, polycarbonate, polyethylene terephthalate,polyamide, polyvinyle chloride, etc. and mixtures thereof.

Biodegradable polymers for use in the present invention include, forexample, polylactic acid (PLA), polyglycolic acid (PGA),polycaprolactone (PCL), polyanhydrides, poly(β-hydroxybutyrate),polydioxanone, poly(DTH iminocarbonate), polypropylene fumarate, etc.copolymers thereof and mixtures thereof.

In certain embodiments, the 3D tubular scaffolds incorporate one or morebiomolecules, e.g., by being coated onto the 3D tubular scaffolds, bybeing extruded together with the polymer when manufacturing the tubularscaffold, or be intermixing the biomolecules with the polymers prior tomanufacture. A biomolecule can be a protein, peptide, glycoaminoglycan,a naturally occurring compound or polymer, a therapeutic agent or acombination thereof. Examples of naturally occurring compound or polymerare collagen, laminin, or fibronectin. Therapeutic agents include butare not limited to, antibiotics, hormones, growth factors, anti-tumoragents, anti-fungal agents, anti-viral agents, pain medications,anti-histamines, anti-inflammatory agents, anti-infective, wound healingagents, anti-proliferative agent, wound sealants, cellular attractants,cytokines and the like. A therapeutic agent is anything that whenapplied to cell would benefit human health.

In certain embodiments, the 3D tubular scaffolds incorporateantibiotics. Antibiotics are chemotherapeutic agents that inhibit orabolish the growth of micro-organisms, such as bacteria, fungi, orprotozoans. Examples of common antibiotics are penicillin andstreptomycin. Other known antibiotics are amikacin, gentamicin,kanamycin, neomycin, netilmicin, tobramycin, paromomycin, geldanamycin,herbimycin, loracarbef, ertapenem, doripenem, imipenem/cilastatin,meropenem, cefadroxil, cefazolin, cefalotin or cefalothin, cefalexin,cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime,cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefdinir, cefepime,teicoplanin, vancomycin, azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, telithromycin,spectinomycin, aztreonam, amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,nafcillin, piperacillin, ticarcillin, bacitracin, colistin, polymyxin B,ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, mafenide,prontosil, sulfacetamide, slfamethizole, slfanilimide, sulfasalazine,sulfisoxazole, trimethoprim, trimethoprim-sulfamethoxazole,demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline,arsphenamine, chloramphenicol, clindamycin, lincoamycin, ethambutol,fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid,metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide,quinupristin/dalfopristin, rifampin or rifampicin and tinidazole.

In certain embodiments, the 3D tubular scaffolds incorporate hormones. Ahormone is a chemical messenger that carries a signal from one cell (orgroup of cells) to another via the blood. Examples of hormones aremelatonin, serotonin, thyroxine, triiodothyronine, epinephrine,norepinephrine, dopamine, antimullerian hormone, adiponectin,adrenocorticotropic hormone, angiotensinogen and angiotensin,antidiuretic hormone, atrial-natriuretic peptide, calcitonin,cholecystokinin, corticotropin-releasing hormone, erythropoietin,follicle-stimulating hormone, gastrin, ghrelin, glucagon,gonadotropin-releasing hormone, growth hormone-releasing hormone, humanchorionic gonadotropin, human placental lactogen, growth hormone,inhibin, insulin, insulin-like growth factor, leptin, luteinizinghormone, melanocyte stimulating hormone, oxytocin, parathyroid hormone,prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulatinghormone, thyrotropin-releasing hormone, cortisol, aldosterone,testosterone, dehydroepiandrosterone, androstenedione,dihydrotestosterone, estradiol, estrone, estriol, progesterone,calcitriol, calcidiol, prostaglandins, leukotrienes, prostacyclin,thromboxane, prolactin releasing hormone, lipotropin, brain natriureticpeptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide,renin, and enkephalin,

In certain embodiments, the 3D tubular scaffolds incorporate growthfactors. Growth factor refers to a naturally occurring protein capableof stimulating cellular proliferation and cellular differentiation.Examples are transforming growth factor beta (TGF-β), granulocyte-colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), nerve growth factor (NGF), neurotrophins,platelet-derived growth factor (PDGF), erythropoietin (EPO),thrombopoietin (TPO), myostatin (GDF-8), growth differentiation factor-9(GDF9), acidic fibroblast growth factor (aFGF or FGF-1), basicfibroblast growth factor (bFGF or FGF-2), epidermal growth factor (EGF),and hepatocyte growth factor (HGF).

In certain embodiments, the 3D tubular scaffolds incorporate antitumors.Antitumors or antineoplastics are drugs that inhibit and combat thedevelopment of tumors. Examples are actinomycin (e.g., actinomycin-D),anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin), bleomycin,plicamycin, Paclitaxel and mitomycin.

In certain embodiments, the 3D tubular scaffolds incorporate anti-fungalagents. An anti-fungal agent is medication used to treat fungalinfections. Examples are natamycin, rimocidin, filipin, nystatin,amphotericin B, miconazole, ketoconazole, clotrimazole, econazole,bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole,sertaconazole, sulconazole, tioconazole, fluconazole, itraconazole,isavuconazole, ravuconazole, posaconazole, voriconazole, terconazole,terbinafine, amorolfine, naftifine, butenafine, anidulafungin,caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine,griseofulvin, gentian violet, haloprogin, tolnaftate, undecylenic acid,tea tree oil, citronella oil, lemon grass, orange oil, palmarosa oil,patchouli, lemon myrtle, neem seed oil, coconut oil, zinc, and selenium.

In certain embodiments, the 3D tubular scaffolds incorporate antiviralagents. Antiviral agents are a class of medication used specifically fortreating viral infections. Examples are abacavir, aciclovir, acyclovir,adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla,brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine,docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,entry inhibitors (fusion inhibitor), famciclovir, fomivirsen,fosamprenavir, foscarnet, fosfonet; ganciclovir, gardasil, ibacitabine,immunovir, idoxuridine, imiquimod, indinavir, inosine, integraseinhibitor, interferon type III, interferon type II, interferon type I,lamivudine, lopinavir, loviride, MK-0518 (raltegravir), maraviroc,moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues,oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin,protease inhibitor (pharmacology), reverse transcriptase inhibitor,ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergisticenhancer (antiretroviral), tenofovir, tenofovir disoproxil, tipranavir,trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, and zidovudine.

In certain embodiments, the 3D tubular scaffolds incorporate painmedications. Pain medications or analgesics (colloquially known as apainkiller) are members of the diverse group of drugs used to relievepain. Examples are paracetamoUacetaminophen, nonsteroidalanti-inflammatory drugs (NSAIDs), COX-2 inhibitors (e.g., rofecoxib andcelecoxib), morphine, codeine, oxycodone, hydrocodone, diamorphine,pethidine, tramadol, buprenorphine, tricyclic antidepressants (e.g.,amitriptyline), carbamazepine, gabapentin and pregabalin.

In certain embodiments, the 3D tubular scaffolds incorporateantihistamines. An antihistamine is a histamine antagonist that servesto reduce or eliminate effects mediated by histamine, an endogenouschemical mediator released during allergic reactions. Examples are H1antihistamine, aceprometazine, alimemazine, astemizole, azatadine,azelastine, benadryl, brompheniramine, chlorcyclizine, chloropyramine,chlorphenamine, phenylpropanolamine, cinnarizine, clemastine, cyclizine,cyproheptadine, dexbrompheniramine, dexchlorpheniramine,diphenhydramine, doxylamine, ebastine, emedastine, epinastine,fexofenadine, histamine antagonist (e.g., cimetidine, ranitidine, andfamotidine; ABT-239, thioperamide, clobenpropit, impromidine,thioperamide, cromoglicate, nedocromil), hydroxyzine, ketotifen,levocabastine, mebhydrolin, mepyramine, mthapyrilene, methdilazine,olopatadine, pheniramine, phenyltoloxamine, resporal, semprex-D,sominex, talastine, terfenadine, and triprolidine.

In certain embodiments, the 3D tubular scaffolds incorporateanti-inflammatory agents. Anti-inflammatory agent refers to a substancethat reduces inflammation. Examples are corticosteroids, ibuprofen,diclofenac and naproxen, helenalin, salicylic acid, capsaicin, andomega-3 fatty acids.

In certain embodiments, the 3D tubular scaffolds incorporateanti-infective agents. Anti-infective agent is any agent capable ofpreventing or counteracting infection. It could be divided into severalgroups. Anthelminthics is one group of anti-infective agents comprisingof albendazole, levamisole, mebendazole, niclosamide, praziquantel, andpyrantel. Another group is antifilarials, such as diethylcarbamazine,ivermectin, suramin sodium, antischistosomals and antitrematodemedicine, oxamniquine, praziquantel, and triclabendazole. Another groupis the antibacterials, which can be further subdivided. The beta lactammedicines are amoxicillin, ampicillin, benzathine benzylpenicillin,benzylpenicillin, cefazolin, cefixime, ceftazidime, ceftriaxone,cloxacillin, co-amoxiclav, imipenem/cilastatin, phenoxymethylpenicillin,and procaine benzylpenicillin. Other antibacterials are azithromycin,chloramphenicol, ciprofloxacin, clindamycin, co-trimoxazole,doxycycline, erythromycin, gentamicin, metronidazole, nitrofurantoin,spectinomycin, sulfadiazine, trimethoprim, and vancomycin. Examples ofantileprosy medicines are clofazimine, dapsone, and rifampicin. Examplesof antituberculosis medicines are amikacin, p-aminosalicylic acid,capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, kanamycin,ofloxacin, pyrazinamide, rifampicin, and streptomycin. Examples ofantifungal medicines are amphotericin B, clotrimazole, fluconazole,flucytosine, griseofulvin, nnystatin, potassium iodide. Antiviral agentsare also anti-infective agents. An example of a antiherpes medicine isacyclovir. Examples of antiretrovirals are nucleoside/nucleotide reversetranscriptase inhibitors. Other examples are abacavir, didanosine,emtricitabine, lamivudine, stavudine, tenofovir disoproxil fumarate,zidovudine, non-nucleoside reverse transcriptase inhibitors, efavirenz,nevirapine, protease inhibitors, indinavir, lopinavir+ritonavir,nelfinavir, ritonavir, saquinavir and ribavirin. Examples ofantiprotozoal medicines are antiamoebic and antigiardiasis medicinessuch as diloxanide, metronidazole; antileishmaniasis medicines such asamphotericin B, meglumine antimoniate, pentamidine; antimalarialmedicines, such as amodiaquine, artemether, artemether+lumefantrine,artesunate, chloroquine, doxycycline, mefloquine, primaquine, quinine,sulfadoxine+pyrimethamine, chloroquine, and proguanil. Antipneumocytosisand antioxoplasmosis medicines are pentamindine, pyrimethamine,sulfamethoxazole+trimethoprim. Antitrypanosomal medicines areeflornithine, melarsoprol, pentamidine, suramin sodium, benznidazole,and nifurtimox. Antimigraine medicines, acetylsalicylic acid,paracetamol, and propranolol.

In certain embodiments, the 3D tubular scaffolds incorporate woundhealing agents. Wound healing agents facilitate the body's naturalprocess of regenerating dermal and epidermal tissue. Examples arefibrin, fibronectin, collagen, serotonin, bradykinin, prostaglandins,prostacyclins, thromboxane, histamine, neuropeptides, kinins,collagenases, plasminogen activator, zinc-dependent metalloproteinases,lactic acid, glycosaminoglycans, proteoglycans, glycoproteins,glycosaminoglycans (GAGs), elastin, growth factors (PDGF, TGF-β), nitricoxide, and matrix metalloproteinases, Examples of wound sealants areplatelet gel and fibrin.

In certain embodiments, the 3D tubular scaffolds incorporateanti-proliferative agents. Anti-proliferative agents prevent tissue fromgrowth, such as to prevent restenosis (recurrent narrowing) of coronary,scar tissue formation, etc. an example of anti-proliferative agent isPaclitaxel. Applying a paclitaxel coating in a coronary stent limitsrestenosis or the growth of neointima (scar tissue).

In certain embodiments, the 3D tubular scaffolds incorporate cellularattractants. Cellular attractants or chemotaxic agents are chemicals ormolecules in the environment that are sensed by bodily cells, bacteria,and other single-cell or multicellular organisms affecting theirmovements. Examples are amino acids, formyl peptides [e.g.,N-formylmethionyl-leucyl-phenylalanine (fMLF or fMLP in references],complement 3a (C3a) and complement 5a (C5a), chemokines (e.g., IL-8);leukotrienes [e.g., leukotriene B4 (LTB4)].

In certain embodiments, the 3D tubular scaffolds incorporate cytokines.Cytokines are group of proteins and peptides that are signalingcompounds produced by animal cells to communicate with one another.Cytokines can be divided into several families. Examples are the fouralpha-helix bundle family with three subfamilies: the IL-2 subfamily[e.g., erythropoietin (EPO) and thrombopoietin (THPO)], the interferon(IFN) subfamily, the IL-10 subfamily. Other examples are the IL-1 family(e.g., IL-1 and IL-18), the IL-17 family, chemokines, immunoglobulin(Ig) superfamily, haemopoietic growth factor (type I) family, Interferon(type 2) family, tumor necrosis factors (TNF) (type 3) family, seventransmembrane helix family, and transforming growth factor betasuperfamily.

In certain embodiments, the surface or partial surface of the 3D tubularscaffolds can be further treated by a physiochemical mean, a chemicalmean, a coating mean, or a combination thereof to improve cellularattachment.

In certain embodiments, the surface of the 3D tubular scaffold can befurther treated with surface modification techniques pertaining tophysiochemical means known in the art to improve the surface property ofthe tubular scaffold for better cellular attachment, by treatment with,e.g., plasma or glow discharge.

Additionally, the surface of the 3D s tubular scaffolds can be furthersurface treated by chemical means, particularly with acids or bases. Ina specific embodiment, the tubular scaffold is treated with H₂SO₄, HNO₃,HCl, H₃PO₄, H₂CrO₄, or a combination thereof. In a specific embodiment,the tubular scaffold is treated with NaOH, KOH, Ba(OH)₂, CsOH, Sr(OH)₂,Ca(OH)₂, LiOH, RbOH, or a combination thereof.

The surface of the 3D tubular scaffolds may also be treated by coatingmeans, in which a substance is applied on the surface that is differentfrom the material of the struts and/or fibers. The substance can becovalently bonded or physically absorbed to the surface of the strutsand/or fibers. Alternatively, the substance can be bonded to the surfaceof the construct through hydrogen bonding, ionic bonding, Van der Waalsforce or a combination thereof. To increase the stability of thebiological molecular coating, the coating can be crosslinked usingvarious crosslinking technologies, such as chemical crosslinking,radiation, thermal treatment, or a combination thereof, etc. Further,the crosslinking can take place in a vacuum at an elevated temperatureabove room temperature. The radiation used for crosslinking can bee-beam radiation, gamma radiation, ultraviolet radiation, or acombination thereof.

The coating substance can be a protein, peptide, glycoaminoglycan, anaturally occurring substance, an inorganic substance, a therapeuticagent, or a combination thereof.

The surface of the 3D tubular scaffolds can be further coated withbiological molecules or naturally occurring compound or polymer, suchas, but not limited to, collagen (type I, II, III, IV, V, IV, etc),fibronectin, laminin, or other extracellular matrix molecules. Examplesof extracellular matrix molecules are heparan sulfate, chondroitinsulfate, keratan sulfates, hyaluronic acid, elastin, hemicellulose,pectin, and extensin. The biological molecules are either covalentlybonded to the surface, or physically absorbed to the surface of thetubular scaffolds.

The surface of the 3D tubular scaffolds can be further surface coatedwith a synthetic polymer, such as, polyvinyl alcohol, polyethyleneglycol, polyvinyl polypyrrolidone, poly(L-lactide), polylysine, etc.

The surface of the 3D tubular scaffolds can also be coated with organicsubstance, such as gelatin, chitosan, polyacrylic acid, polyethyleneglycol, polyvinyl alcohol, polyvinylpyrrilidone or a combinationthereof.

Alternatively, the 3D s tubular scaffolds may be coated with aninorganic material, such as calcium phosphate, TiO₂, Al₂O₃, or acombination thereof.

In a specific embodiment, the 3D tubular scaffolds are coated with acomposite coating of two or more organic materials, such as, gelatin andchitosan, polyacrylic acid and polyethylene glycol, polyvinyl alcoholand polyvinylpyrilidone, etc.

The 3D tubular scaffolds may also be coated with a composite ofinorganic materials, such as calcium phosphate and TiO₂, calciumphosphate and Al₂O₃, etc.

The 3D tubular scaffolds may also be coated with a composite coating ofa combination of inorganic and organic materials, such as, calciumphosphate/collagen, calcium phosphate/gelatin, calciumphosphate/polyethylene glycol, etc.

The 3D tubular scaffolds of the present invention may also be usedtogether with fabrics. The fabrics can be attached either to the outersurface or the inner surface or to both surfaces of the tubularscaffolds.

The fabrics for attaching to 3D tubular scaffolds can be either woven ornon-woven fabrics or both.

The fabrics can be prefabricated or directly fabricated on the outersurface of the 3D tubular scaffolds. The direct fabrication method willincluded, but not limit to, an electrospinning fabrication method inwhich the rotation shaft with the 3D scaffolds still on serves as thefiber collector.

The Apparatus and Methods of Manufacture of a 3D Tubular Scaffold

As illustrated in FIG. 2, a 4^(th) axis, containing a computercontrolled rotation shaft, is added to an XYZ position system. This 4axis fabrication system also has a materials delivery system mounted onthe XYZ position system. In preferred embodiments, the materialsdelivery system is a polymer melt extrusion system which is able todirectly deposit polymer material in a hot melt filament form, or asolution delivery system which can deliver viscous polymer solutions onto the computer controlled rotation shaft. The computer controlledrotation shaft driven by a server motor can be programmed precisely torotation, stop and rotate back and forth at desired speeds. The rotatingshaft can be equipped with a heating element or hosted in a temperaturecontrolled environment to control the softness and viscosity of thedelivered material so that the materials will exhibit the desiredproperties. For example, when using a polymer solution, the deliveredmaterials can adhere better to each other and maintain a certain shape.The hot melt filament will bond to previously extruded filaments whenthey meet, therefore eliminating the need to use glue. For a givenpolymer, a set of preferred combinations of flow rate, head speed andmelt chamber temperature can be established in order to producesufficient adhesion between extrudate of adjacent layers at theircross-points while maintaining vertical channels between them, toprovide interconnectivity throughout the entire structure.

The 3D tubular scaffolds may be fabricated using several methods, e.g.,via a layer by layer fabrication technique or layer by layer assemblingtechnique. In such an embodiment, each layer of 3D tubular scaffold ispre-fabricated using suitable polymer processing techniques according tothe structure design.

The layers of the 3D tubular scaffold are then assembled together byputting several layers of the tubular scaffolds inside one another. Eachlayer of 3D tubular scaffold may have a different structure and may alsobe longer than the desired length of the final product. When the lengthof the construct is longer than the final desired product, the finalproduct can be cut into the desired length using a mechanical device,such as a knife or a laser beam. One or more final 3D tubular scaffoldsmay be cut from a single assembled long construct.

Hot melt polymers may be used in the present invention for fabricatingporous 3D tubular structures. Polymer pellets/particles/beads may beused directly without the need to fabricate polymeric filament first. Torealize the direct use of polymer hot melt, the apparatus of the presentinvention has an extruder which is mounted on a dispensing arm. Theextruder is equipped with a delivery mechanism, such as compressed air,a plunger, a extrusion screw, or a combination of the above, to forcethe molten polymer through a nozzle, which is attached to the extruder.The apparatus also comprises a rotary rod positioned underneath thenozzle. The extruded polymer thin filament deposits onto the rotatingrotary rod according to a designed moving pathway in a layer by layerfashion. The rotary rod rotates to allow the polymer to deposit onsurfaces of the rod while the extruder moves along the Y-axis, therebyforming a tubular shape, with polymer melt depositing in a desiredpattern. Multiple layers of polymer melt can be deposited on the 4^(th)rotation axis to form a thicker wall with a porous structure. A porousstructure thus can be obtained when the solidified polymer is removedfrom the rotation shaft. In order to allow for greater control overporosity, pore size and structure, either the dispensing arm, rotary rodor both move along a longitudinal axis. In addition to this longitudinalmovement, the speed of the rotation of the rotary rod and the speed ofthe longitudinal movement of the dispensing arm and/or the rotary rodcan also aid in the control of pore size tubular e of the 3D tubularscaffold.

In certain embodiments, the 3D scaffolds of the present invention may bemanufactured using a 4-axis RP system, in which a dispensing device ismounted on the XYZ dispensing arm is used to dispense the polymersolution. The dispensing device is equipped with a delivery mechanism,such as compressed air, a plunger or a combination of the two, to forcethe polymer solution through a nozzle or a syringe needle which isattached to the dispensing device. The apparatus also comprises a rotaryrod positioned underneath the nozzle. The extruded polymer solutionstream deposits onto the rotating rotary rod in a layer by layerfashion. The rotary rod is equipped with a temperature control mechanismso that the polymer solution deposited onto the rotary rod is quicklysolidified such as frozen, therefore, the deposited polymer solution canbe kept at its deposited location and maintain certain size and shape.The freeze mechanism can be a temperature controlled freeze chamber or arotary rod equipped with a cooling coil inside the shaft of the rod.Similar to the polymer melt deposition described above, the rotary rodrotates to allow the polymer solution to deposit on surfaces of the rodwhile the dispensing device moves along the Y-axies, thereby forming the3D tubular scaffold with frozen polymer solution deposited in aparticular pattern. Multiple layers of frozen polymer solution can bedeposited on the 4^(th) rotation axis to form a thicker tubular wallporous structure. When finishing the deposition process, the frozenpolymer structure, along with the rotary rod, is removed from the systemand put into a freeze-drying chamber. The solvent of the polymersolution is then removed through a sublime process leaving behind asolid polymer structure. When finishing the solvent removing process, aporous tubular structure can be obtained when the dried polymer isremoved from the rotary rod. In order to allow for greater control overporosity, pore size and structure, either the dispensing arm, rotary rodor both move along a longitudinal axis. In addition to this longitudinalmovement, the speed of the rotation of the rotary rod and the speed ofthe longitudinal movement tubular of the dispensing arm and/or therotary rod can also aid in the control of pore size of the 3D scaffold.

The present invention can use any type of thermal plastic polymerpellets, beads, particles, which are suitable for extrusion, injectionmolding, or forming solution with a solvent, as well as composites oftwo or more different thermal plastic polymer blends, inorganicparticle/thermal plastic composites.

The materials used in this thermal extrusion apparatus can be a singletype of thermal plastic polymer or a blend of two or more polymers in apreformed form. They can be a physical mixture ofpellets/beads/particles of two or more thermoplastic polymers in apremixed mixture form. Additionally, the materials can be a physicalmixture of inorganic particles and thermoplastic polymerparticles/pellets/beads. Additionally, the materials can be a suspensioninorganic particles suspended in a polymer solution. The materials canalso be a solution from polymers and small molecules. In preferredembodiments, a micro single screw extruder is used, as it will result ina more homogeneous dispersion of inorganic particle in polymer matrix.

The material can also include a low molecular weight substance, such asa therapeutic agent.

In certain embodiments, the polymer melt extrusion mechanism or solutiondispensing device is equipped with a on/off switch or a regulator whichcontrols the extrusion rate of the polymer melt or solution. The switchcan be a pressure regulator or a pressure valve that switch thecompressed air on/off when compressed gas is used for extrusion ofpolymer melt. When the switch is on, the polymer melt or solution willbe extruded though the nozzle tip by the air pressure inside the meltchamber. The switch can also be a electronic switch which turns on/offthe motor that controls the rotation movement of the screw in a microsingle-screw extruder or in a screw driven plunger.

The diameter of the extruded/dispensed polymer filament can becontrolled by the inner diameter of the extrusion/dispensing nozzle andthe extrusion/dispensing speed.

Applications of the 3D Tubular Scaffolds

The 3D tubular scaffolds of the present invention may be used, e.g., forregeneration of blood vessels. In one embodiment, the 3D tubularscaffold may be seeded and cultured with endothelia cells in vitro, andthen implanted to replace the damaged blood vessel. In anotherembodiment, a small tubular scaffold may be seeded and cultured withendothelial cells, then inserted into a larger tubular scaffold witchhas been seeded and cultured with smooth muscle cells. Afterco-culturing in vitro for certain period of time, the blood vesselconstruct then can be implanted to replace the damaged blood vessel.

The 3D tubular scaffolds of the present invention may be used asvascular stents, which when placed and expanded in a plagued andnarrowed segment of a blood vessel, the stent will keep the blood vesselopen for easy blood flow.

Additionally, the 3D tubular scaffolds of the present invention may beused, e.g., to fabricate porous tubular structure for large bone defectrepair or bone tissue engineering, as trabecular bone is a tubularstructure with bone marrow resides in the center portion of the tubularbone. With the porous wall structure, stem cells or osteoblasts may beseeded and culture to produced regenerated bone tissue.

The 3D tubular scaffolds of the present invention may be used asesophagus stents, which, when placed and expanded in a narrowed segmentof a esophagus, will keep the esophagus open for easy food flow intostomach. Such an application will be advantageous for late stageesophagus cancer patients.

Similarly, the 3D tubular scaffolds of the present invention may also beused as stents designed for the intestines, the bile conduct, urinarytract, etc.

The 3D tubular scaffolds of the present invention may also be used,e.g., for nerve regeneration. The porous scaffolds can be used asperiphery nerve conduit to guide the damaged nerve to regenerate. Inthis application, the two ends of the periphery nerve can be insertedinto the 3D tubular structure and guide the nerve to grow and reunite.

1. An apparatus for manufacturing a three-dimensional tubular scaffoldcomprising: (i) a three-axis XYZ system connected to a base; (ii) adispensing system connected to the XYZ system; (iii) a nozzle connectedto the dispensing system; and (iv) a fourth axis system comprising arotary rod connected to the base under the nozzle, wherein either therotary rod, the nozzle or both are capable of moving along alongitudinal axis.
 2. The apparatus of claim 1, wherein the dispensingsystem is a polymer extruder.
 3. The apparatus of claim 1, wherein thedispensing system is a syringe.
 4. The apparatus of claim 1, wherein thedispensing system is a pump.
 5. The apparatus of claim 1, furthercomprising a temperature control.
 6. A method of making athree-dimensional tubular scaffold comprising: (i) adding a polymer intothe dispensing system of the apparatus of claim 1; and (ii) dispensingthe polymer through the nozzle onto the rotary rod.
 7. A method claim 6,further comprising the steps of drying the polymer and removing thedried polymer from the scaffold.
 8. A three-dimensional tubular scaffoldcomprising struts and/or fibers joined in a porous three-dimensionalpattern, the scaffold having an average pore size from about 1 to about2000 microns, wherein the scaffold is tubular.
 9. The three-dimensionaltubular scaffold of claim 8, wherein the struts and or fibers comprise apolymer.
 10. The three-dimensional tubular scaffold of claim 9, whereinthe polymer is biodegradable.
 11. The three-dimensional tubular scaffoldof claim 9, wherein the polymer is non-biodegradable.
 12. Thethree-dimensional tubular scaffold of claim 8, wherein the scaffoldfurther comprises at least one therapeutic agent.
 13. Thethree-dimensional tubular scaffold of claim 8, wherein the struts and orfibers comprise an inorganic polymer composite.
 14. Thethree-dimensional tubular scaffolds of claim 8, wherein the strutscomprise a coating on the surface.
 15. The three-dimensional tubularscaffolds of claim 8, wherein the scaffolds comprise fabric attached tothe outer surface of the scaffold, the inner surface of the scaffold, orto both surfaces of the scaffold.
 16. The three-dimensional tubularscaffolds of claim 15, wherein the fabric is woven or non-woven.
 17. Thethree-dimensional tubular scaffolds of claim 15, wherein the fabric isprefabricated and attached to the scaffold or fabricated directly ontothe surface of the scaffold.